Reagent and process for the identification and counting of biological cells

Abstract

The invention relates to a reagent and a process for the identification and counting of biological cells in a sample. This reagent comprises a cell lysing agent selected from at least one detergent in a concentration capable of specifically lysing a given type of cells in the sample, and a stain capable of marking the intracellular nucleic acids of the remaining unlysed cells. Application in particular for the identification and counting of cells using an automated analysis system based on flow cytometry.

1. A process for the identification and counting of biological cells in a sample comprising erythrocytes, the process comprising the following operations in a single stage:
 simultaneously lysing the erythrocytes by mixing and incubating the sample with a cell lysing agent comprising at least one detergent in a concentration capable of specifically lysing erythrocytes in the sample at least one membrane fixing agent, and a membrane penetration agent selected from the group consisting of an ionophore compound of the protonophore type, an ionophore of the antibiotic type, and a mixture of ionophores, and 
 staining intracellular nucleic acids of unlysed cells, 
 to provide a mixture of lysed erythrocytes and unlysed cells having stained intracellular nucleic acids, and 
 identifying and counting the unlysed cells by flow cytometry using at least two measuring parameters selected from the group consisting of resistive volume, axial luminous diffraction, axial luminous transmission, orthogonal light scattering, and fluorescence. 
 
     
     
       2. The process according to  claim 1 , wherein the cell lysing agent comprises at least one ionic and/or non-ionic detergent in a concentration capable of lysing erythrocytes. 
     
     
       3. The process according to  claim 1 , wherein the detergent is selected from the group consisting of:
 primary amines, amine acetates and hydrochlorides, quaternary ammonium salts, and trimethylethyl ammonium bromide; 
 amides of substituted diamines, diethanolamino-propylamine or diethylaminopropylamide, amides of cyclised diethylenetriamine; 
 alkylaryl sulfonates, petroleum sulfonates, sulfonated glycerides; 
 cholamides, sulfobetaines; 
 alkyl glycosides, saponins; 
 polyoxyethylene ethers and sorbitans, and polyglycol ethers. 
 
     
     
       4. The process according to  claim 1 , wherein the stain is a fluorescent type stain. 
     
     
       5. The process according to  claim 1 , wherein the stain is capable of combining specifically with intracellular ribonucleic acid of said unlysed remaining cells whereby the fluorescence of said stain is enhanced. 
     
     
       6. The process according to  claim 1 , wherein the stain is selected from the group consisting of:
 thiazole orange or 1-methyl-4-[(3-methyl-2-(3H)-benzothiazolylidene)methyl]quinolinium p-tosylate, 
 thiazole blue, 
 4-[(3-methyl-2-(3H)-benzothiazolyl-idene)methyl]-1-[3-(trimethylammonium)propyl]quinolinium diiodide, 
 3,3′-dimethyloxacarbocyanine iodide or 3-methyl-2-[3-(3-methyl-2(3H)-benzothiazolylidene-1-propenyl]benzoxazolium iodide, 
 thioflavine T, 
 the stains SYTO® and TOTO® (TM Molecular Probes), 
 ethidium bromide, 
 propidium iodide, 
 acridine orange, 
 coriphosphine O, 
 auramine O, 
 the stains 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperizinyl)-2,5′-bi-1H-benzimidazole trihydrochloride hydrate and 2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperizinyl)-2,5′-bi-1H-benzimidazole trihydrochloride, 
 4′,6-diamino-2-phenylindole dihydrochloride (DAPI), 
 4′,6-diimidazolin-2-yl)-2-phenylindole dihydrocholoride (DIPI), 
 7-aminoactinomycin D, 
 actinomycin D, and 
 LDS 751 (2-(4-(4-dimethylaminophenyl)-1,3-butadienyl)-3-ethylbenzothiazolium perchlorate). 
 
     
     
       7. The process according to  claim 1 , wherein the at least one membrane fixing agent is present in a concentration of 0.1% to 10% (w/v). 
     
     
       8. The process according to  claim 7 , wherein the membrane fixing agent comprises at least one alcohol or at least one aldehyde selected from the group consisting of paraformaldehyde and glutaraldehyde, or a mixture of at least one said alcohol and at least one said aldehyde. 
     
     
       9. The process according to  claim 1 , wherein the cell lysing agent also comprises at least one compound selected from the group consisting of a complexing agent, an inorganic salt and a buffer system. 
     
     
       10. The process according to  claim 1 , wherein the resistive volume measurement is carried out by application of any one of a continuous current, a pulsed current, or an alternating current. 
     
     
       11. The process according to  claim 1 , wherein the axial luminous diffraction parameter is at least one parameter selected from the group consisting of small angle diffraction and large angle diffraction. 
     
     
       12. The process according to  claim 1 , wherein the unlysed cells having stained intracellular nucleic acids are either mature or immature, normal or abnormal cells. 
     
     
       13. The process according to  claim 1 , further comprising classifying the unlysed cells by means of a multidimensional analysis software program, with or without the use of a software or other neuronal technique. 
     
     
       14. The process according to  claim 1 , wherein the sample is a sample of human or animal blood. 
     
     
       15. The process according to  claim 1 , wherein the sample is a sample of biological fluid or a suspension of cells of human or animal origin.